Integrative Real-Time NMR and X-ray Crystallography Reveal Prostaglandin D₂ Metabolism

Understanding the metabolic pathways and kinetics of prostaglandins is essential for elucidating their biological functions and therapeutic potential. Prostaglandin D₂ (PGD₂), a somnogen in the brain, undergoes nonenzymatic conversion into J-series prostaglandins, including 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂). PGD₂ is possibly transported by lipocalin-type prostaglandin D synthase (L-PGDS), which may also influence its metabolism. However, the kinetics of PGD₂ metabolism, particularly in the context of the PGD₂-L-PGDS complex, remains poorly understood. In this study, we investigated the effects of L-PGDS on the dehydration reaction of PGD₂ in an aqueous buffer using real-time NMR spectroscopy, complemented by UV-visible absorption spectroscopy. In the absence of L-PGDS, 15d-PGJ₂ was formed over several tens of hours via the transient accumulation of prostaglandin J₂ and 15-deoxy-Δ^12,14-prostaglandin D₂ as intermediates. In contrast, the PGD₂-L-PGDS complex converted to a L-PGDS-15d-PGJ₂ complex, without exhibiting detectable reaction intermediates or byproducts, at a time scale of 3 h. We also determined the crystal structure of the L-PGDS-15d-PGJ₂ complex, demonstrating that the covalent bond is formed between Cys65 of L-PGDS and the carbon atom at the C9 position of 15d-PGJ₂. These results, combined with the fact that L-PGDS is present in excess relative to PGD₂ and 15d-PGJ₂ in the arachnoid membrane, suggest that most PGD₂ exists in the L-PGDS-bound form and that 15d-PGJ₂ generated through dehydration is rapidly and effectively sequestered by L-PGDS. Thus, L-PGDS may function as a scavenger for 15d-PGJ₂, mitigating its potential deleterious effects in the arachnoid membrane. This real-time NMR-based approach provides a useful platform for studying the metabolism behavior of other prostaglandins under physiologically relevant conditions.